US20130171674A1 - Method for quantifying the amount of cholesterol in high-density lipoprotein 3 - Google Patents

Method for quantifying the amount of cholesterol in high-density lipoprotein 3 Download PDF

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US20130171674A1
US20130171674A1 US13/811,562 US201113811562A US2013171674A1 US 20130171674 A1 US20130171674 A1 US 20130171674A1 US 201113811562 A US201113811562 A US 201113811562A US 2013171674 A1 US2013171674 A1 US 2013171674A1
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surfactant
polyoxyethylene
cholesterol
hdl3
ether
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Maiko Higuchi
Yasuki Itoh
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Denka Co Ltd
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Denka Seiken Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/08—Sphingolipids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the present invention relates to a method for quantifying cholesterol in high-density lipoprotein 3 (which may be hereinafter referred to as “HDL3”) (cholesterol in HDL3 may be hereinafter referred to as “HDL3 cholesterol”).
  • HDL3 high-density lipoprotein 3
  • HDL3 cholesterol cholesterol in HDL3
  • HDL high-density lipoprotein
  • a predetermined lower limit is regarded as an indication of dislipidemia, and the value is known to be useful as an index of arteriosclerosis.
  • HDL is constituted by apoprotein, phospholipid, cholesterol and triglyceride.
  • HDL can be divided into subfractions based on the difference in the content of apolipoprotein E among apoproteins in HDL, and HDLs having higher contents of apoE are defined as apoE-rich HDL.
  • HDL has been conventionally studied as a whole, but each of the subfractions HDL2 and HDL3 is now known to have unique functions. It is clinically known that CETP deficiency prevents cholesterol transport from HDL to LDL and IDL, leading to an increase in the HDL cholesterol level.
  • the HDL increased by CETP deficiency is HDL2.
  • HDL2 is said to have an antiarteriosclerotic action. It is also said that CETP deficiency causes an increase in apoE-rich HDL, and that, since apoE-rich HDL has a strong cholesterol-drawing ability and antiplatelet action, it is a good HDL.
  • a decrease in the hepatic lipase activity prevents conversion of HDL3 to HDL2, resulting in an increase in HDL3. It is suggested that increased HDL3 leads to increased incidence rates of coronary artery diseases. In view of such tendencies, it is expected that measurement of each HDL subfraction may contribute to judgment of whether or not a patient is suffering from an arteriosclerotic disease and of the cause of the disease. Further, at present, in view of these functions of HDL subfractions, manufacturers are developing therapeutic agents that inhibit the function of CETP, decrease the LDL cholesterol level, and increase the HDL cholesterol level.
  • HDL subfractions examples include ultracentrifugation, high-performance liquid chromatography (HPLC), HDL3 precipitation (Patent Document 1) and NMR.
  • HDL3 precipitation is a method wherein a reagent containing a divalent metal ion and dextran sulfate is used to aggregate lipoproteins other than HDL3, and HDL3 in the supernatant portion is recovered by centrifugation and measured using an automatic analyzer.
  • NMR which is a method wherein the number of particles of lipoprotein is measured by magnetic resonance, is not commonly employed since the method requires special equipment.
  • Patent Document 2 There is another method for analyzing an HDL subfraction (Patent Document 2). Although this method enables measurement with a general purpose automatic analyzer, the method employs a method wherein a surfactant is used to prevent an enzyme from acting on lipoproteins other than HDL3. Therefore, since the HDL3 reaction is allowed to proceed in the presence of the lipoproteins other than the lipoprotein of interest, the measurement might be influenced by such lipoproteins or, in cases where the prevention is not sufficient, the lipoproteins other than HDL3 might be undesirably measured together.
  • An object of the present invention is to provide a method that enables quantification of HDL3 in a test sample without requiring a laborious operation.
  • the present inventors intensively studied to discover that phospholipase and sphingomyelinase act on lipoprotein but hardly act on HDL3.
  • the present inventors then inferred that HDL3 cholesterol in a test sample can be quantified by allowing phospholipase or sphingomyelinase to act on the test sample to eliminate cholesterol and then quantifying cholesterol in the remaining HDL3.
  • the present inventors then experimentally confirmed that this is possible, thereby completing the present invention.
  • the present invention provides a method for quantifying cholesterol in high-density lipoprotein 3, the method comprising:
  • Step 1 wherein phospholipase and/or sphingomyelinase is/are allowed to act on a test sample to transfer cholesterol to the outside of the reaction system;
  • Step 2 wherein cholesterol remaining in the reaction system is quantified.
  • HDL3 cholesterol in a test sample can be specifically quantified with an automatic analyzer without requirement of a laborious operation such as ultracentrifugation or pretreatment. Further, quantification of the HDL2 cholesterol level can also be carried out by subtracting the HDL3 cholesterol level from the total HDL cholesterol level obtained by a conventional method for quantifying the total HDL cholesterol in a test sample.
  • FIG. 1 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent D in Step 2.
  • FIG. 2 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent G in Step 2.
  • FIG. 3 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent H in Step 2.
  • FIG. 4 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent I in Step 2.
  • FIG. 5 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent K in Step 2.
  • FIG. 6 shows a diagram illustrating a result of Examples of the present invention in terms of changes in the absorbance of each fraction after addition of Reagent L in Step 2.
  • test sample to be subjected to the method of the present invention is not restricted as long as HDL3 cholesterol in the sample can be quantified, and the test sample is preferably serum or blood plasma, or a dilution thereof. Serum or a dilution thereof is especially preferred.
  • phospholipase and/or sphingomyelinase (these may be hereinafter referred to as the “phospholipase and/or the like”) is/are allowed to act on a test sample.
  • the phospholipase is not restricted as long as it acts on phosphatidyl choline.
  • Phospholipase A, phospholipase C and phospholipase D are preferred, and phospholipase C and phospholipase D are especially preferred. Since the phospholipase and the like are commercially available, commercially available products may be preferably used.
  • Each of the phospholipase and/or the like may be used alone, or two or more types of the phospholipase and/or the like may be used in combination.
  • the final concentration of the phospholipase and/or the like is preferably about 0.1 to about 100 U/mL, more preferably about 0.2 to about 50 U/mL.
  • HDL3 is hardly affected, while lipoproteins other than HDL3 are affected, resulting in transfer of cholesterol of the lipoproteins to the outside of the reaction system.
  • Step 1 of the method of the present invention cholesterol is transferred to the outside of the reaction system by the action(s) of the phospholipase and/or the like.
  • the term “transferred to the outside of the reaction system” herein means that cholesterol and esters thereof are eliminated or protected such that the cholesterol and esters thereof are not involved in the later steps.
  • the term “elimination” herein means that cholesterol of lipoprotein in a test sample is degraded such that the cholesterol does not affect the reaction for measurement of cholesterol in a later step.
  • Examples of the method for eliminating lipoprotein cholesterol include a method wherein cholesterol esterase and cholesterol oxidase are allowed to act on the cholesterol, followed by decomposition of the produced hydrogen peroxide into water and oxygen using catalase. Alternatively, a hydrogen donor may be reacted with the produced hydrogen peroxide using peroxidase to convert the hydrogen peroxide to a colorless quinone.
  • the method for eliminating lipoprotein cholesterol is not restricted to these. The method of elimination of cholesterol per se is known in the art, and is also described concretely in Examples below.
  • protection means to protect lipoprotein in a test sample such that the lipoprotein does not react upon cholesterol measurement in a later step.
  • examples of the method of protection of lipoprotein include, but are not limited to, a method wherein a surfactant is used to specifically protect each lipoprotein such that cholesterol esterase and cholesterol oxidase do not act on the lipoprotein.
  • Step 1 by preliminarily adding an enzyme system and a surfactant for transferring cholesterol to the outside of the reaction system at the same time, both steps can be carried out at the same time as a single step.
  • the concentration (the concentration means the final concentration unless otherwise specified in the present specification) of cholesterol esterase is preferably about 0.1 to about 10.0 U/mL, more preferably about 0.2 to about 2.0 U/mL.
  • the concentration of cholesterol oxidase is preferably about 0.05 to about 10.0 U/mL, more preferably about 0.1 to about 1.0 U/mL.
  • the cholesterol esterase is not restricted as long as it acts on ester-type cholesterol, and examples of the cholesterol esterase which may be used include commercially available products such as cholesterol esterase (CEBP, CEN) manufactured by Asahi Kasei Corporation and cholesterol esterase (COE-311, COE-312) manufactured by Toyobo Co., Ltd. Further, the cholesterol oxidase is not restricted as long as it acts on free cholesterol, and examples of the cholesterol oxidase which may be used include commercially available products such as cholesterol oxidase (CONII) manufactured by Asahi Kasei Corporation and cholesterol oxidase (COO-311, COO-321, COO-331) manufactured by Toyobo Co., Ltd.
  • CONII cholesterol oxidase
  • COO-321, COO-331 cholesterol oxidase
  • the concentration of peroxidase is preferably about 2.0 to about 5.0 U/mL, more preferably about 3.0 to about 4.0 U/mL. In cases where a compound for conversion into a colorless quinone is used, the concentration of the compound is preferably about 0.4 to about 0.8 mmol/L.
  • the solution is preferably Good's, Tris, phosphate or glycine buffer solution, and is preferably a Good's buffer such as bis(2-hydroxyethyl)iminotris(hydroxyethyl)methane (Bis-Tris), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-ethanesulfonic acid) sesqui sodium salt monohydrate (PIPES 1.5Na), 3-morpholinopropanesulfonic acid (MOPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) or piperazine-1,4-bis(2-hydroxy-3-propa
  • Bis-Tris bis(2-hydroxyethyl)iminotris(hydroxyethyl)methane
  • PPES
  • the reaction temperature in Step 1 is preferably about 25 to about 40° C., more preferably 35 to 38° C., most preferably 37° C.
  • the reaction time is not restricted, and is usually about 2 to about 10 minutes.
  • Step 1 can be carried out in the absence of a surfactant, Step 1 is preferably carried out in the coexistence of a surfactant that reacts with lipoproteins other than HDL or HDL3 in view of accurate quantification of HDL3 cholesterol.
  • the term “react” when used for a surfactant in the present invention, the term means that the surfactant leads lipoprotein to the outside of the reaction system, making an enzyme act easily, or means to protect lipoprotein such that an enzyme cannot act on the lipoprotein.
  • surfactant that reacts with lipoproteins other than HDL include, but are not limited to, anionic surfactants such as polyoxyethylene-polyoxypropylene condensates, polyoxyethylene alkyl ether sodium sulfate amide nonion, polyoxyethylene nonylphenyl ether and amide nonion; and nonionic surfactants such as polyoxyethylene polycyclic phenyl ether having an HLB value of 14 to 17.
  • anionic surfactants such as polyoxyethylene-polyoxypropylene condensates, polyoxyethylene alkyl ether sodium sulfate amide nonion, polyoxyethylene nonylphenyl ether and amide nonion
  • nonionic surfactants such as polyoxyethylene polycyclic phenyl ether having an HLB value of 14 to 17.
  • surfactant examples include Pluronic, Pluronic P123 (ADEKA Corporation), Pluronic F88 (ADEKA Corporation), Levenol WX (Kao Corporation), Nonion HS-220 (NOF Corporation), Nymid MT-215 (NOF Corporation), Newcol-723 (NOF Corporation), Newcol-2614 (Nippon Nyukazai Co, Ltd.) and Newcol-714 (Nippon Nyukazai Co, Ltd.).
  • surfactant that reacts with lipoproteins other than HDL3 include, but are not limited to, nonionic surfactants such as polyoxyethylene distyrene-modified phenyl ether; anionic surfactants such as polyoxyethylene-polyoxypropylene condensates, amide ether sulfate and polyoxyethylene-stearylamine; amphoteric surfactants such as coconut oil fatty acid-amidopropyldimethyl-aminoacetic acid betaine, alkyl dimethyl-aminoacetic acid betaine and lauryl betaine; and cationic surfactants such as lauryl trimethyl ammonium chloride.
  • nonionic surfactants such as polyoxyethylene distyrene-modified phenyl ether
  • anionic surfactants such as polyoxyethylene-polyoxypropylene condensates, amide ether sulfate and polyoxyethylene-stearylamine
  • amphoteric surfactants such as coconut oil fatty acid-amid
  • examples of the nonionic surfactants include polyoxyethylene distyrene-modified phenyl ether Emulgen A500 (trade name; manufactured by Kao Corporation; company names hereinafter represent names of manufacturers, and all names described together with company names hereinafter represent trade names);
  • examples of the anionic surfactants include polyoxyethylene-polyoxypropylene condensates Pluronic F127 (ADEKA Corporation), Pluronic F68 (ADEKA Corporation) and Pluronic P103 (ADEKA Corporation), amide ether sulfate Sunamide CF-10 (NOF Corporation) and polyoxyethylene-stearylamine Nymeen S210 (NOF Corporation);
  • examples of the amphoteric surfactants include a coconut oil fatty acid-amidopropyldimethyl-aminoacetic acid betaine Nissan Anon BDF-SF (NOF Corporation), alkyl dimethyl-aminoacetic acid betaine Nissan Anon BF (NOF Corporation) and lauryl betaine Amphitol 24B (Kao Corporation);
  • the concentration of the surfactant that reacts with lipoproteins other than HDL3 is preferably 0.01 to 5.0% by weight, more preferably about 0.03 to about 3.0% by weight.
  • reaction conditions reaction temperature, time, buffer and the like are as described above.
  • Step 2 cholesterol remaining in the reaction system is quantified.
  • the “surfactant that reacts with at least HDL3” includes a surfactant that specifically reacts with HDL3, surfactant that specifically reacts with HDL (that is, reacts with HDL2 and HDL3) and surfactant that reacts with all lipoproteins.
  • examples of the surfactant that specifically reacts with HDL3 include nonionic surfactants such as polyoxyethylene distyrene-modified phenyl ether Emulgen A90 (Kao Corporation), polyoxyethylene lauryl ether Emulgen 120 (Kao Corporation) and p-isooctyl polyoxyethylene phenol formaldehyde polymer Triton-WR-1339 (Nacalai Tesque); amphoteric surfactants such as lauryl dimethyl-aminoacetic acid betaine Nissan Anon BL-SF (NOF Corporation) and polyoxyethylene lauryl ether Persoft NK-100 (NOF Corporation); and cationic surfactants such as fatty acid series phosphoric acid ester ADEKA COL PS-440E (ADEKA Corporation).
  • nonionic surfactants such as polyoxyethylene distyrene-modified phenyl ether Emulgen A90 (Kao Corporation), polyoxyethylene lauryl ether Emulgen 120 (Kao Corporation) and
  • Examples of the surfactant that specifically reacts with HDL include nonionic surfactants having an HLB of 11 to 14 such as polyoxyethylene distyrene-modified phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyalkylene alkyl ether, polyoxyethylene polycyclic phenyl ether and polyoxyethylene cumyl phenyl ether; and imidazoline-type amphoteric surfactants.
  • Emulgen A60 Kerat Corporation
  • Emulgen B66 Keratin B66
  • Emulgen LS110 Keratin CMP-11
  • Newcol-710 Nippon Nyukazai Co, Ltd.
  • Newcol-610 Nippon Nyukazai Co, Ltd.
  • Newcol-2609 Nippon Nyukazai Co, Ltd.
  • Nissan Anon GLM-R-LV NOF Corporation
  • surfactant that specifically reacts with all lipoproteins examples include nonionic surfactants such as polyoxyethylene lauryl ether, polyoxyethylene alkyl ether and polyoxyethylene alkyl phenyl ether.
  • Anionic surfactants such as lauryl alcohol alkoxylate and sodium polyoxyethylene-alkyl phenyl ether sulfate may also be used.
  • Emulgen 707 Keratin 707
  • Emulgen 909 Keratin 909
  • Emulgen 108 Keratin 108
  • Nymeen L207 NOF Corporation
  • Adekatol LB83 ADKA Corporation
  • Adekatol LB103 ADKA Corporation
  • Newcol-707 Neippon Nyukazai Co, Ltd.
  • the concentration of the surfactant is preferably 0.01 to 5.0% (w/v), more preferably 0.05 to 2.0% (w/v).
  • Step 2 cholesterol is quantified by the reaction of such surfactants.
  • the surfactant used is different between Step 1 and Step 2.
  • Methods of quantification per se of cholesterol are well known, and any of the well known methods may be used. A concrete description is also given in Examples below. For example, ester-type cholesterol in lipoprotein is hydrolyzed with cholesterol esterase to produce free cholesterol and a fatty acid, and the produced free cholesterol and free cholesterol inherently existing in lipoprotein are converted using cholesterol oxidase to generate cholestenone and hydrogen peroxide. A quinone pigment is then formed in the presence of peroxidase, and quantified.
  • Examples of compounds that generate a quinone pigment include HDAOS (N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline), DAOS (N-ethyl-N(-2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt) or TOOS (N-ethyl-N(-2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt dihydrate) and 4-aminoantipyrine, but the compounds are not restricted as long as the combination allows generation of a quinone pigment.
  • the cholesterol esterase and cholesterol oxidase used in Step 1 may be used as they are in Step 2 without further addition.
  • the concentration of the compound for generation of a quinone pigment is preferably about 0.5 to about 2.0 mmol/L in the case of HDAOS, or 0.1 to 2.0 mmol/L in the case of 4-aminoantipyrine.
  • the concentration of peroxidase is preferably 0.4 to 5.0 U/mL.
  • a catalase inhibitor sodium azide is used by addition to the reaction liquid in Step 2.
  • the concentration of sodium azide in this case is usually about 0.1 g/L to about 1.0 g/L.
  • reaction temperature, time, buffer, pH and the like may be the same as the reaction conditions for Step 1 described above.
  • the HDL2 cholesterol level in the test sample by subtracting the HDL3 cholesterol level obtained by Step 1 and Step 2 from the HDL cholesterol level in the test sample. Since methods for determining the HDL cholesterol level in a test sample are well known (e.g., JP 2001-103998 A) and kits for such methods are commercially available, the quantification can be easily carried out using these.
  • Reagent A and Reagent B having the compositions described below were prepared, and reagents were prepared by adding various surfactants to Reagent A to a concentration of 0.1% (w/v) or 1.0% (w/v).
  • Reagent A containing the various surfactants described below was mixed with Reagent B at a ratio of 1:3. Cholesterol in each of the HDL2 fraction and the HDL3 fraction was reacted with the resulting mixture, and the final absorbances at a main wavelength of 700 nm and a sub-wavelength of 600 nm were measured and compared.
  • HDL2 fraction Fractionation was carried out to obtain the HDL2 fraction and the HDL3 fraction as follows.
  • a test sample containing HDL that is, serum was subjected to ultracentrifugation using a solution with sodium chloride and sodium bromide such that separation occurs at a density at the border between HDL2 and HDL3 (1.125), and each resulting fraction was collected.
  • Table 1 below shows surfactants with which the ratio of HDL2/HDL3 was not more than 0.75 and the ratios of CM-IDL/HDL3 and LDL/HDL3 were not more than 0.75. These surfactants were determined to be surfactants that react with HDL3.
  • Table 2 shows surfactants with which the ratio of HDL2/HDL3 was not less than 1.25 and the ratios of CM-IDL/HDL2 and LDL/HDL2 were not less than 1.25. These surfactants were determined to be surfactants that react with lipoproteins other than HDL3.
  • Table 3 shows surfactants with which the ratio of HDL2/HDL3 was between 0.75 and 1.25 and the ratios of CM-IDL/HDL2, LDL/HDL2, CM-IDL/HDL3 and LDL/HDL3 were not more than 0.75. These surfactants were determined to be surfactants that react with HDL.
  • Table 4 shows surfactants with which the ratio of HDL2/HDL3 was between 0.75 and 1.25 and the ratios of CM-IDL/HDL2, LDL/HDL2, CM-IDL/HDL3 and LDL/HDL3 were not less than 0.75. These surfactants were determined to be surfactants that react with all lipoproteins.
  • Table 5 shows surfactants that could not be grouped into any of the above categories. These surfactants were determined to be surfactants that react with lipoproteins other than HDL.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL
  • Emulgen A90, Emulgen 120, Nissan Anon BL-SF, Triton WR-1339, Persoft NK-100 and ADEKA COL PS-440E were surfactants that specifically react with HDL3.
  • Emulgen A500, Nissan Anon BDF-SF, Nissan Anon BF, Nymeen S210, Pluronic P103, Kohtamin 24P, Sunamide CF-10, Amphitol 24B, Pluronic F68 and Pluronic F127 were surfactants that specifically react with lipoproteins other than HDL3.
  • Emulgen B66, Emulgen A60, Emulgen LS110, Nowol 610, Newcol 2609, Newcol-CMP-11, Nissan Anon GLM-RLV and Newcol-710 were surfactants that specifically react with HDL.
  • Emulgen 108, Emulgen 707, Newcol 707, Adekatol LB83, Adekatol LB103 and Emulgen 909 were surfactants that specifically react with all lipoproteins.
  • Newcol-714, Newcol-723, Newcol-2614, Nymeen S215, Pluronic P123, Levenol WX, Nymid MT-215, Nonion HS220 and Pluronic F88 were surfactants that specifically react with lipoproteins other than HDL.
  • CM-LDL fraction Fractionation by ultracentrifugation was carried out to obtain the CM-LDL fraction, HDL2 fraction and HDL3 fraction, and each fraction was reacted with Reagent E prepared by adding phospholipase D (PLDP) to Reagent A used in Reference Example 1.
  • Reagent D described below was further added to the reaction solution to perform measurement.
  • 150 ⁇ L of Reagent E was added to 2 ⁇ L of serum, and the reaction was allowed to proceed for 5 minutes with warming, followed by addition of Reagent D to the reaction solution and additional 5 minutes of reaction with warming
  • the absorbances at a main wavelength of 700 nm and a sub-wavelength of 600 nm were measured.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Phospholipase D 5.0 U/mL
  • FIG. 1 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent D. Specific reaction with HDL3 can be seen.
  • Reagent F was prepared by adding a surfactant and sphingomyelinase to Reagent A used in Reference Example 1, and Reagent G was prepared for the step of measuring HDL3 in the product obtained by reaction with Reagent F.
  • the compositions of these reagents were as shown below. Fractionation was carried out by ultracentrifugation to obtain the fraction from CM to IDL, LDL fraction, HDL2 fraction and HDL3 fraction, and the obtained fractions were subjected to measurement. The procedure for measurement with the reagents was the same as in Example 1. The absorbance was measured at each measurement time.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Pluronic F68 0.03 w/v % Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Sphingomyelinase 0.5 U/mL
  • FIG. 2 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent G. Specific reaction with HDL3 can be seen.
  • the absorbance was measured in the same manner as in Example 2 using Reagent H which was prepared with the same composition as that of Reagent E used in Example 1 except that phospholipase C was used as the phospholipase and a surfactant was added.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Pluronic F68 0.03 w/v % Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Phospholipase C 5.0 U/mL
  • FIG. 3 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent H. Specific reaction with HDL3 can be seen.
  • the absorbance was measured in the same manner as in Example 2 using Reagent I which was prepared with the same composition as that of Reagent H used in Example 3 except that phospholipase D (PLDP) was used as the phospholipase and a surfactant was added.
  • PLDP phospholipase D
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Pluronic F68 0.03 w/v % Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Phospholipase D 5.0 U/mL
  • FIG. 4 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent I. Specific reaction with HDL3 can be seen.
  • the absorbance was measured in the same manner as in Example 2 except that Reagent J and Reagent K having the compositions described below were used.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Pluronic P103 0.03 w/v % Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Phospholipase D 5.0 U/mL
  • FIG. 5 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent K. Specific reaction with HDL3 can be seen.
  • the absorbance was measured in the same manner as in Example 2 except that Reagent J used in Example 5 and Reagent L having the composition described below were used.
  • FIG. 6 shows the result in terms of changes in the absorbance with time of each fraction after addition of Reagent L. Specific reaction with HDL3 can be seen.
  • Reagent M was prepared and used in combination with Reagent D to measure HDL-C (C represents cholesterol; the same applies hereinafter) in the test sample. From this value, the value obtained by measuring HDL3-C using Reagent I and Reagent D in Example 4 was subtracted, to calculate HDL2-C. The values of HDL-C, HDL3-C and HDL2-C in each sample were as shown in Table 6.
  • BES buffer (pH 7.0) 100 mmol/L HDAOS 0.7 mmol/L Pluronic P88 0.1 w/v % Catalase 600 U/L Cholesterol oxidase 1.4 U/mL Cholesterol esterase 0.8 U/mL Phospholipase D 5.0 U/mL

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JP7153460B2 (ja) * 2018-03-30 2022-10-14 シスメックス株式会社 リポタンパク質の取り込み能を測定する方法及び試薬
CN109813921B (zh) * 2019-02-25 2022-04-19 安徽大千生物工程有限公司 基于多克隆抗体制备的hdl3免疫比浊法检测试剂盒及其制备使用方法
CN109837325B (zh) * 2019-03-15 2022-08-12 安徽大千生物工程有限公司 一种基于修饰鞘磷脂酶优化的hdl3比色法检测试剂盒及其制备使用方法
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